ABSTRACT
Amphibians such as Xenopus laevis and Ambystoma mexicanum are capable of whole structure regeneration. However, transcriptional control over these events is not well understood. Here, we investigate the role of histone deacetylase (HDAC) enzymes in regeneration using HDAC inhibitors. The class I/II HDAC inhibitor valproic acid (VPA) inhibits tail regeneration in embryos of the anuran amphibian Xenopus laevis, confirming a recent report by others (Tseng et al., 2011). This inhibition correlates with a sixfold reduction in endogenous HDAC activity. VPA also inhibited tail regeneration in post-refractory stage Xenopus larvae and larvae of the urodele A. mexicanum (axolotl). Furthermore, Xenopus limb regeneration was also significantly impaired by post-amputation treatment with VPA, suggesting a general requirement for HDAC activity in the process of appendage regeneration in amphibians. The most potent inhibition of tail regeneration was observed following treatment with VPA during the wound healing, pre-blastema phase. A second HDAC inhibitor, sodium butyrate, was also shown to inhibit tail regeneration. While both VPA and sodium butyrate are reported to block sodium channel function as well as HDACs, regeneration was not inhibited by valpromide, an analogue of VPA that lacks HDAC inhibition but retains sodium channel blocking activity. Finally, although VPA is a known teratogen, we show that neither tailbud nor limb bud development are affected by exposure to this compound. We conclude that histone deacetylation is specifically required for the earliest events in appendage regeneration in amphibians, and suggest that this may act as a switch to trigger re-expression of developmental genes.
Subject(s)
Amphibians/growth & development , Extremities/physiology , Histone Deacetylases/metabolism , Regeneration/physiology , Tail/physiology , Xenopus laevis/physiology , Ambystoma mexicanum/growth & development , Ambystoma mexicanum/metabolism , Amphibians/genetics , Amphibians/metabolism , Animals , Butyrates/pharmacology , Gene Expression Regulation, Developmental/drug effects , Gene Expression Regulation, Developmental/genetics , Histone Deacetylase Inhibitors/pharmacology , Histone Deacetylases/genetics , Larva/drug effects , Larva/genetics , Larva/growth & development , Larva/metabolism , Regeneration/drug effects , Regeneration/genetics , Sodium Channel Blockers/pharmacology , Sodium Channels/genetics , Sodium Channels/metabolism , Valproic Acid/analogs & derivatives , Valproic Acid/pharmacology , Wound Healing/drug effects , Wound Healing/genetics , Wound Healing/physiologyABSTRACT
The design and fabrication of a membrane-integrated microfluidic cell culture device (five layers,≤500 µm total thickness) developed for high resolution microscopy is reported here. The multi-layer device was constructed to enable membrane separated cell culture for tissue mimetic in vitro model applications and pharmacodynamic evaluation studies. The microdevice was developed via a unique combination of low profile fluidic interconnect design, substrate transfer methodology, and wet silane bonding. To demonstrate the unique high resolution imaging capability of this device, we used oil immersion microscopy to image stained nuclei and mitochondria in primary hepatocytes adhered to the incorporated membrane.
